Take home messages

• Reduced levels of sensitivity to several DMI fungicides was detected in Western Australia in the net blotch pathogen
  • net form of net blotch from 2013
  • spot form of net blotch from 2016
• All isolates from the Northern region tested up to date remained sensitive to fungicides
• Control of both NFNB and SFNB is best achieved by
  • Use of resistant varieties
  • Reduce inoculum load by managing stubble and good farm hygiene
  • Only spray if necessary – limit applications
  • Use best fungicide management practices such as lowest effective dose & rotating fungicide groups
  • Treat seed to limit spread of seed-borne NFNB isolates

Background

The two forms of net blotch in barley, net form of net blotch (NFNB) and spot form of net blotch (SFNB) are caused by one of two forms of Pyrenophora teres (P. teres). NFNB is caused by P. teres f. teres (Ptt) and SFNB is caused by P. teres f. maculata (Ptm).

Symptoms of SFNB are characterised by dark circular or elliptic brown patches surrounded by a yellow chlorotic region, whereas NFNB are characterised by net like dark brown necrotic lesions.  The two forms are morphologically identical and can only be distinguished by symptoms and molecular characterisation (McLean et al. 2009).

Control of both NFNB and SFNB is best achieved by the use of resistant varieties, reducing inoculum load by stubble management, crop rotation, good farm hygiene and fungicide application.  In recent years, resistance to particular fungicides have been observed in both pathogens.

Fungicides are essential in maintaining healthy crops and are applied routinely in most barley crops.  The choice of fungicide is determined by registration, efficacy, availability and price.  The Australian market is dominated by a small number of systemic actives from a single mode of action (MOA) group, these being the DMI’s or Group 3 triazole fungicides.  Fungicide efficacy varies with disease. When conditions are favourable for disease development, a repeat application may be required for effective disease control.

The efficacy of some fungicides have been impacted by the development of resistance in pathogens.  Most modern fungicides have a single-site mode of action and act on a specific biochemical pathway in the target fungal pathogen.  Repeated use of fungicides with the same mode of action, selects for isolates in the fungal population with reduced sensitivity to the fungicide.  Multi-site fungicides are less prone to resistance development in target pathogens and play an important role in the strategy against the development of fungicide resistance (FRAG-UK 2018).

In Australia, fungicide resistance in barley pathogens have been identified to date in powdery mildew, spot form of net blotch (SFNB) and net form of net blotch (NFNB).  Pathogen populations are routinely monitored by the Centre for Crop and Disease Management (CCDM) Fungicide Resistance Group (FRG) based at Curtin University in Western Australia (WA).  Disease samples are collected Australia wide and submitted to CCDM for analysis to determine resistance levels in a range of pathogens to the fungicides registered for control.  More information is available on the FRG website.

Demethylase inhibitor (DMI) fungicide resistance in NFNB

NFNB isolates with reduced levels of sensitivity to several DMI fungicides were detected in WA from 2013 onwards via discriminatory dose screening (Table 1).  Strains were classified as either sensitive (S), moderately resistant (MR), or highly resistant (HR) based on 50% effective concentrations (EC₅₀) to the Group 3 compounds prothioconazole, tebuconazole and epoxiconazole (Table 2).  EC₅₀ ofsensitive isolates were derived from samples collected by DPIRD and from field trips, at eight locations: Amelup, Badgingarra, Esperance, Jerramungup, Kalannie, Merredin, Pithara, and Wongan Hills. Moderately resistant strains were first found in samples from a bait trial in the Great Southern (Kojonup) region in 2013, and subsequently detected throughout the wheatbelt and Great Southern regions in samples collected by collaborators from Bakers Hill, Dandaragan, Mount Barker, Scaddan, Tenterden and West Arthur, as well as from a bait trial in Beverley.  Highly resistant isolates were found in the Esperance (Scaddan), southern wheatbelt (West Arthur) and central wheatbelt (Dandaragan) regions from 2017 onwards.

Table 1. Number of NFNB isolates tested in each year showing proportion that are sensitive, moderately resistant or highly resistant to Group 3 (DMI) fungicides. ^aFrom 2018 season onwards, strains were reisolated and tested only where molecular detection had previously confirmed presence of resistance mutations in samples.

Table 2. Mean effective concentration 50 (EC₅₀) in µg/mL of thirteen Group 3 moderately resistant isolates, three highly resistant isolates, and 8 sensitive NFNB isolates collected 1996–2012.  Resistance factors (fold number difference between EC₅₀ values from resistant isolates and average of sensitive isolates) are given in brackets.  Cultures were grown at different concentration ranges of the fungicides tebuconazole, epoxiconazole and prothioconazole.

Analysis of the target gene for the Group 3 fungicides, called Cyp51A, revealed the presence of one mutation in MR and HR isolates. In MR isolates, there was a single fungicide target gene with the mutation.  However, in HR isolates, up to 10 additional fungicide target genes carrying the mutation were detected.  These extra mutated target genes are probably involved in the over-production of the Group 3 fungicide target which could explain the higher levels of resistance of HR isolates to the fungicides tested.

DMI fungicide resistance in SFNB

SFNB isolates with reduced levels of sensitivity to several DMI fungicides were detected in Western Australian from 2016 onwards via discriminatory dose screening (Table 3).  In vitro testing sorted strains into sensitive (S), moderately resistant (MR), and highly resistant (HR) groups based on EC₅₀ concentrations to the Group 3 compounds prothioconazole, tebuconazole and epoxiconazole.  The MR and HR groups showed a similar level of reduced sensitivity to epoxiconazole (Table 4).  EC₅₀ ofsensitive isolates were derived from samples collected by DPIRD and from field trips, at the following locations: Albany, Amelup, Boscabel, Durham Ox, Esperance, Irvingdale, Jerramungup, Kebaringup, Lumeah, Mooree, Morawa, Munglinup and Muresk, as well as from bait trials at Kojonup and Meckering.  Moderately resistant strains were detected in samples collected in field trips in the Esperance region (Gibson, Coomalbidgup and Cascade) from 2016 onwards, and highly resistant isolates were found in samples sent by collaborators from the Great Southern (South Stirling and Wellstead) and Esperance (Dalyup) regions from 2017 onwards.

Table 3. Number of SFNB isolates tested in each year showing proportion that are sensitive, moderately resistant or highly resistant to Group 3 (DMI) fungicides.  ^aFrom 2018 season onwards, strains were reisolated and tested only where molecular detection had previously confirmed presence of resistance mutations in samples.

Table 4. Mean effective concentration 50 (EC₅₀) in µg/ml of five Group 3 moderately resistant isolates, fifteen highly resistant isolates, and the mean of a reference population of 23 sensitive SFNB isolates collected between 1995 and 2013.  Resistance factors (fold number difference between EC₅₀ values from resistant isolates and average of sensitive isolates) are given in brackets. Cultures were grown at different concentration ranges of the fungicides tebuconazole, epoxiconazole and prothioconazole.

Analysis of thegroup 3 fungicide target Cyp51A, revealed the presence in MR and HR isolates of two different mutations that were not observed in sensitive isolates.  In MR isolates, the mutation was a small fragment of DNA that was inserted in the fungicide target gene.  This small fragment of new DNA was found at three different positions and its effect was the over-production of the fungicide target.  The presence of more fungicide target requires an increase in the amount of fungicide necessary to kill the MR isolates.

The small DNA fragment was also found in HR isolates but at a different position, together with another mutation, F489L.  This latter mutation has been previously observed in the closely related pathogen P. teres f. teres, the causative agent of net form of net blotch (NFNB), where it has been correlated with reduced sensitivity to a range of DMI fungicides (Mair et al. 2016).

Net blotch in the Northern Region (NR)

Both forms of the net blotch pathogen occurs regularly in the Northern Region and isolates are collected annually, however in 2018 disease pressure was very low due to the dry conditions.  A total of 12 NFNB isolates were collected in the NR between 2009 and 2017, mostly from baiting trials. None of these isolates showed reduced sensitivity to fungicides applied.  As NFNB is a seed-borne pathogen, the use of an appropriate seed-dressing will aid in limiting the spread of isolates with reduced sensitivity or resistance to fungicides.

Management

In addition to using cultivars with good disease resistance levels, stubble management strategies to reduce disease load, crop rotation and maintaining good farm hygiene, the following chemical management strategies are recommended:

• Only spray if necessary – limit applications
• Choose mixtures with different modes of action (if available). Overuse of fungicides with the same mode of action will speed up fungicide resistance
• Never apply the same Group 3 fungicide twice in a row
• Avoid applying the same mode of action fungicides from the Groups 7 and 11 twice
• Incorporate the use of seed dressing (Group 7), in-furrow (Group 11) and foliar products containing fungicide mixtures from different chemical groups (such as 3 (DMI), 7 (SDHI) and 11 (Qol)) - in combination with limited use of propiconazole and no stand-alone tebuconazole use
• Avoid using tebuconazole as a stand-alone product in barley for any disease to avoid indirect fungicide resistance selection
• Ideally use DMI-based mixtures (e.g. Prosaro^Ò containing prothioconazole and tebuconazole) only once, followed by mixtures containing other actives (preferably from groups 7 or 11)
• If resistance is present, or suspected, avoid or minimise use of that mode of action - this will only further select for resistance
• Do not exceed label rates

References

FRAG-UK, 2018. Fungicide Resistance Management in Cereals.

GRDC fact sheet, 2012. Barley Powdery Mildew.

GRDC Media Release, 2018. Fungicide resistance found in barley spot form of net blotch.

GRDC Media Release, 2018. Research uncovers new changes in fungicide resistance in WA barley.

Mair WJ, Deng W, Mullins JGL, West S, Wang P, Besharat N, Ellwood SR, Oliver RP & Lopez-Ruiz FJ, (2016)  Demethylase Inhibitor Fungicide Resistance in Pyrenophora teres f. sp. teres associated with Target Site Modification and Inducible Overexpression of Cyp51. Front. Microbiol. 7:1279. doi: 10.3389/fmicb.2016.01279

McLean MS, Howlett BJ & Hollaway GJ, (2009) Epidemiology and control of spot form of net blotch (Pyrenophora teres f. maculata) on barley: a review. Crop and Pasture Science 60,303-315

Acknowledgements

The research undertaken as part of this project is made possible by the significant contributions of growers through both trial cooperation and the support of the GRDC, the authors would like to thank them for their continued support.

Contact details

Lislé Snyman
DAF QLD
Hermitage Research Facility, 604 Yangan Rd, Warwick, Qld
Ph: 07 4542 6761
Email: lisle.snyman@daf.qld.gov.au

Fran Lopez-Ruiz
Curtin University, Centre for Crop and Disease Management
School of Molecular and Life Sciences
Perth WA 6845
Ph: 08 9266 3061
Email: fran.lopezruiz@curtin.edu.au